Screening for weak monoclonal antibodies in hybridoma technology

As the interest in weak-affinity antibodies has been widened by their introduction to various analytical techniques such as HPLC, capillary electrophoresis and biosensors, there has been a need for new screening/monitoring methods. In this study, weak-affinity chromatography was adopted to screen/monitor directly for monoclonal antibodies in ascites. Monoclonal antibodies against a carbohydrate antigen (maltohexaose) were used to evaluate this approach. In short, malthohexaose was immobilized on an HPLC support in such a configuration to allow, during HPLC, retardation of weak monoclonal antibodies. Based on the retention, the affinity or the avidity, as determined by the presence of multiple binding of the monoclonal antibody towards antigen, can be estimated. In this way it is possible to select clones of hybridomas that produce desired weak monoclonal antibodies. Adjustments in temperature (10-20 degrees C) were used to moderate the retention and hence affinity of the weak monoclonal antibodies during chromatography

Development of monoclonal antibodies against creatine kinase MB2

An increase of creatine kinase MB (CKMB) in serum has long been used as a marker for acute myocardial infarction (AMI). It is important with an early diagnosis and since the amount of the CKMB2 isoform rises above reference levels much earlier than the total creatine kinase amount, quantification of CKMB isoforms could be a feasible alternative for early analysis. The two CKMB isoforms differ by only one C-terminal lysine residue, which makes it difficult to separate one from the other. To overcome this problem, monoclonal antibodies were produced using unique peptides as antigen in hybridoma technology. Two peptides with 16 and 15 amino acids corresponding to the C-terminal end of the M-subunits of CKMB2 and CKMB1 respectively, were conjugated to keyhole limpet hemocyanin and used as antigens. Sixteen different monoclonal antibodies to these peptides were obtained and characterized. Their specificity was analyzed by immunoassay and 10 of the antibodies showed cross-reactive binding to creatine kinase. Surface plasmon resonance based biosensor analysis was used to determine affinity and kinetics towards the peptides and the epitopes of four of the antibodies were studied by means of phage display. Some of these antibodies have binding properties that might qualify them for use in the establishment of procedures allowing early diagnosis of AMI